Means and methods for breaking noncovalent binding interactions between molecules

ABSTRACT

Described are multimeric proteinaceous molecules comprising at least two members that bind each other via a region of noncovalent interaction, wherein a first of the members comprises a conditionally reactive group that, when activated, cleaves a covalent bond within the first member. Cleavage of the covalent bond results in a reduction in the binding strength with which the at least two members bind to each other via the region of noncovalent interaction. The reduction in the binding strength can result in the separation of the members under mild conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 11/883,119, filed Oct. 10, 2007, U.S. Pat. No. 9,079,941 (Jul.14, 2015), which application is a national phase entry under 35 U.S.C.§371 of International Patent Application PCT/NL2006/000038, filed Jan.25, 2006, published in English as International Patent Publication WO2006/080837 A2 on Aug. 3, 2006, which claims the benefit under 35 U.S.C.§119 of European Patent Application Serial No. 5075196.5 filed Jan. 25,2005, the disclosure of each of which is hereby incorporated herein inits entirety by this reference.

TECHNICAL FIELD

This disclosure relates to the field of binding molecules. Thisdisclosure, in particular, relates to means and methods for breakingnoncovalent interactions between members of a multimeric proteinaceousmolecule.

BACKGROUND

Noncovalent interactions are very important, especially in the field ofbiology. Noncovalent bonding holds the two strands of the DNA doublehelix together (hydrogen bonds), folds polypeptides into such secondarystructures as the alpha helix and the beta conformation, enables enzymesto bind to their substrate, enables antibodies to bind to their antigen,enables transcription factors to bind to each other, enablestranscription factors to bind to DNA, enables proteins (e.g., somehormones) to bind to their receptor, and permits the assembly of suchmacromolecular machinery as ribosomes, actin filaments, microtubules andmany more.

There are three principal kinds of noncovalent forces; i.e., ionicinteractions, hydrophobic interactions and hydrogen bonds.

Ionic Interactions as Exemplified by Protein Interactions

At any given pH, proteins have charged groups that may participate inbinding them to each other or to other types of molecules. For example,negatively charged carboxyl groups on aspartic acid (Asp) and glutamicacid (Glu) residues may be attracted by the positively chargedprotonated amino groups on lysine (Lys) and arginine (Arg) residues.

Ionic interactions are highly sensitive to changes in pH. As the pHdrops, H+ bind to the carboxyl groups (COO—) of aspartic acid (Asp) andglutamic acid (Glu), neutralizing their negative charge, and H+ bind tothe unoccupied pair of electrons on the N atom of the amino (NH2) groupsof lysine (Lys) and arginine (Arg), giving them a positive charge. Theresult: Not only does the net charge on the molecule change (it becomesmore positive), but many of the opportunities that its side chain ormain chain groups have for ionic (electrostatic) interactions with othermolecules and ions are altered. As the pH rises, H+ are removed from theCOOH groups of Asp and Glu, giving them a negative charge (COO—), and H+are removed from the NH3+ groups of Lys and Arg, removing their positivecharge. The result: the net charge on the molecule changes (it becomesmore negative) and, again, many of the opportunities its side chain ormain chain groups have for electrostatic interactions with othermolecules or ions are altered.

Ionic interactions are also sensitive to salt concentration. Increasingsalt concentration reduces the strength of ionic binding by providingcompeting ions for the charged residues.

Hydrophobic Interactions as Exemplified by Protein Interactions

The side chains (R groups) of such amino acids as phenylalanine andleucine are nonpolar and, hence, interact poorly with polar moleculeslike water. For this reason, most of the nonpolar residues in globularproteins are directed towards the interior of the molecule, whereas suchpolar groups as aspartic acid and lysine are on the surface exposed tothe solvent. When nonpolar residues are exposed at the surface of twodifferent molecules, it is energetically more favorable for their two“oily” nonpolar surfaces to approach each other closely, displacing thepolar water molecules between them.

The strength of hydrophobic interactions is not appreciably affected bychanges in pH or in salt concentration.

Hydrogen Bonds as Exemplified by Protein Interactions

Hydrogen bonds can form whenever a strongly electronegative atom (e.g.,oxygen, nitrogen) approaches a hydrogen atom, which is covalentlyattached to a second strongly electronegative atom.

Some common examples: between the —C═O group and the H—N— groups ofseparated peptide bonds in proteins (giving rise to the alpha helix andbeta configuration); between —C═O groups and hydroxyl (H—O—) groups inserine and threonine residues and the SH groups of cysteine of proteinsand in sugars.

It is a characteristic of noncovalent interactions that they areindividually weak but collectively strong. All three forms ofnoncovalent interactions are individually weak (on the order of 5kcal/mole) as compared with a covalent bond (with its 90-100 kcal/moleof bond energy). There are types of bonds with an intermediary bondenergy (i.e., between 15 and 70 kcal/mole). For this disclosure, suchtypes of bonds are considered noncovalent if they are by themselvesinsufficient to associate two proteinaceous molecules in a certainenvironment. In other words, noncovalent bonds are those of which asubstantial number of interactions working together are needed to holdstructures together. The limited strength that these interactions dohave requires that the interacting groups can approach each otherclosely (an angstrom or less).

Thus, a multimer comprising two or more members is, in one aspect, saidto be held together by noncovalent bonds if the two or more members arelinked by at least three, and preferably at least five, bonds that eachhave a bond energy of less than 90-100 kcal/mole. A typical cysteinedisulfide bridge linking two protein chains has a bond energy of about65 kcal/mole. However, the strength of this bond is very dependent onthe reducing/oxidizing environment. Thus, for this disclosure, amultimer is said to be held together by noncovalent bonds when the bondsthat link the two or more member chains each have a bond energy of lessthan 65 kcal/mole, and typically less than 20 kcal/mole. Usually, eachof the bonds has a binding energy of around 5 kcal/mole. Thus, two ormore members in a multimer that are held together by noncovalent bondshave a substantial number of noncovalent interactions working togetherto hold the structures together and have a surface topography thatenables substantial areas of the at least two interacting surfaces toapproach each other closely; that is, they must fit each other.

DISCLOSURE

This disclosure utilizes the characteristic that many weak bonds arerequired to keep the structures in a multimeric proteinaceous moleculetogether via a region of non-covalent interaction.

Herein, at least one member involved in the noncovalent interaction inthe multimer is broken up in at least two new members by severing atleast one covalent bond in the at least one member of the multimer. Thecleavage of one covalent bond results in a multimer that has one moremember than the original multimer. Cleavage of two covalent bondsresults in a multimer that has two more members, etc. The cleaving of atleast one covalent bond in the multimer results in a decrease of thenumber of noncovalent interactions per member in the multimer.

This reduction is used to cause dissociation of at least one member fromthe multimer.

The cleavage of a covalent bond is used to reduce the number ofnoncovalent bonds per member in the multimer such that the noncovalentbonds are insufficient to maintain the integrity of the multimer, or thecleavage of a covalent bond is used to alter the conformation of themember in which this modification has occurred and thereby causing therelease of at least one member from the multimer. After release of theat least one member, the resulting members can be monomers, multimers ora combination thereof. Cleavage of a covalent bond in a peptide can beachieved by enzymatic means, chemical means or physical means. Cleavageof a covalent bond as used in the disclosure is preferably chemical orphysical, i.e., preferably non-enzymatic. More preferably, cleavage of abond is chemical- or light-inducible. Further preferred is that acovalent bond is cleaved in a peptide backbone. A covalent bond that ispreferably cleaved is a bond positioned next to a peptide bond (thus, abond on the other side of the nitrogen atom).

As used herein, the term “multimeric proteinaceous molecule” refers to aproteinaceous molecule that contains two or more members that areassociated with each other via a region of noncovalent interaction. Atleast two members are only linked to each other via noncovalentinteractions and not via covalent interactions. The terms “multimericproteinaceous molecule” and “multimeric protein” are interchangeablyused in the description. The multimeric proteinaceous molecule of thisdisclosure typically contains at least one polypeptide.

The term “region of noncovalent interaction” refers to a region wheretwo or more members associate and interact with each other via at leastthree, and preferably at least five, noncovalent bonds. This region ofnoncovalent interaction preferably does not comprise a covalent bondlinking two or more members to each other. It is clear that not allatoms participate in non-covalent interaction in the region. Similarly,if the region is a region where two (poly) peptides are associated, itis not required that all amino acids participate in noncovalentinteraction in the region.

A “monomer” is used herein to refer to a molecule wherein the buildingblocks are still covalently associated with each other when allnoncovalent bonds are broken. More than one monomer in the multimer maybe the same or different from each other.

The term “member” is used herein to refer to an entity of the multimerthat is noncovalently linked to another member of the multimer. Thesetwo members are not linked via a covalent bond. A member is preferably,but not necessarily a proteinaceous molecule. A member is typically, butnot necessarily, a (poly)peptide.

A multimeric proteinaceous molecule of the disclosure is preferably amultimeric protein. A multimeric protein according to the disclosurepreferably comprises a first member comprising a peptide and at least asecond member comprising a (poly)peptide and/or a protein. The peptidepreferably comprises the conditionally reactive group.

A proteinaceous molecule comprises at least two amino acids in peptidiclinkage with each other. It typically contains at least eight aminoacids, or functional equivalents thereof, in peptidic linkage with eachother.

In this disclosure, a polypeptide contains at least 50 amino acids, orfunctional equivalents thereof, that are linked to each other viapeptide bonds. In its unfolded state, the polypeptide is typically alinear molecule but can be (partly) circular. A peptide typicallycontains between four and 49 amino acids that are linked to each othervia peptide bonds. Preferably, a peptide contains from three to 49 aminoacids, preferably from three to 30, more preferably from three to 20amino acids.

A (poly)peptide as used herein can comprise any amino acid or amino acidchain. An amino acid can be a natural or synthetic amino acid such as,for instance, an alpha, beta, or gamma or higher (omega) amino acid,i.e., including one, two, three, or more carbon spacings between aminogroups and carboxylic acids. An amino acid (chain) can be a naturalamino acid (chain) or a synthesized amino acid (chain) or a combinationthereof. A peptide is a natural peptide or a synthesized peptide or acombination thereof. Again, in its unfolded state, a peptide istypically linear, but can be (partly) circular. A peptide typically doesnot have a dominant tertiary structure. It typically accommodates arange of tertiary structures. A peptide as used in the disclosure istypically easily dissolvable in diverse solvents. Such solvents are, forinstance, physiological solutions, such as a physiological sodiumchloride solution.

An antigenic peptide that is a ligand for an MHC molecule typically hasbetween eight and 25 amino acids that are linked via peptide bonds.(Poly)peptides may or may not be modified. Typical modifications includethose as produced by the cellular machinery, such as glycan addition andphosphorylation. However, other types of modification are also withinthe scope of the disclosure.

A functional equivalent of an amino acid is a molecule that can replaceone or multiple amino acids in an amino acid chain. The functionalequivalent is preferably capable of forming bonds with amino acids intwo separate positions such that it can form an internal part of a(poly)peptide or peptidomimetic chain. The functional equivalent doesnot have to have a natural counterpart. Such a functional equivalent canbe incorporated into a peptide or (poly)peptide of the disclosure.

In one aspect of this disclosure, the covalent bond that is cleaved ispreferably a backbone bond that links two amino acids, orderivative/analogue thereof, in one member chain. Cleavage of thecovalent bond is preferably achieved by incorporating a conditionallyreactive group into the member, preferably a peptide member, in which atleast one covalent bond needs to be broken. In this disclosure, the term“conditionally reactive group” is used to refer to a reactive group thatis incorporated into the member. The term “conditionally” is used toreflect that the reactive group can be activated conditionally, i.e., inresponse to a signal or trigger. The number of conditionally reactivegroups can be varied at will. A conditionally reactive group can also beplaced at an exact position in the member, for instance, byincorporating it into a nascent (poly)peptide chain. In one embodiment,the one or more conditionally reactive groups are placed such that thecovalent bond is broken in a region of noncovalent interaction of themember with at least one other member in the multimer. In anotherembodiment, the one or more conditionally reactive groups are placedsuch that the preferred conformation of the ligand (e.g., member) isaltered such that the affinity of the interaction with other members ofthe multimer is reduced.

In one aspect, provided is a multimeric proteinaceous moleculecomprising at least two members that bind each other via a region ofnoncovalent interaction, wherein at least one of at least two memberscomprises a conditionally reactive group that, when activated, cleaves acovalent bond within the member, thereby cleaving the member into atleast two smaller members, which in turn reduces the strength ofnoncovalent interaction in the region. Cleavage of a covalent bond in amember results in more folding freedom for the resulting chains, therebyreducing the strength of any noncovalent interaction that one or more ofthe resulting chains have in the multimer. In a preferred embodiment,the member is cleaved within the region of noncovalent interaction. Thisresults in the highest reduction of the strength of the noncovalentinteraction in the region. The bond that is cleaved by the conditionallyreactive group can be selected by appropriately positioning the reactivegroup in the peptide chain. Cleavage of the member results in peptidechains of a smaller size. It is preferred that the longest of theresulting peptides is at least 20%, and preferably at least 30%, shorterthan the member.

Further provided is a multimeric proteinaceous molecule (a multimer)comprising at least two members that bind each other via a region ofnoncovalent interaction, wherein at least one of at least two memberscomprises a (poly)peptide chain with a conditionally reactive group andwherein, when the reactive group is activated, a covalent bond withinthe member is broken resulting in cleavage of the (poly)peptide into atleast two smaller (poly)peptides, thereby reducing the strength of thenoncovalent interaction.

In a preferred embodiment, the conditionally reactive group comprises alight-sensitive or periodate-sensitive group. In these embodiments, theconditional step that activates the reactive group is either thepresence or absence of light within a defined wavelength range or theexposure of the periodate-sensitive group to periodate. In a preferredembodiment, the light-sensitive reactive group is a UV-sensitive group.The conditionally reactive group is preferably incorporated into a(poly)peptide. In a preferred embodiment, the UV-sensitive groupcomprises 3-amino-3(2-nitrophenyl)propionic acid (Carlos J. Bosques andBarbara Imperiali, Journal of the American Chemical Society 2003, vol.125, pp. 7530-7531). This amino acid is commercially available fromAlpha Aesar and is to be protected with an Fmoc-protecting group inorder to be compatible with solid phase peptide synthesis, orortho-nitrophenyl-glycine (Alvie L. Davis, David R. Smith and Tommy J.McCord, “Synthesis and Microbiological Properties of3-amino-1-hydroxy-2-indolinone and related compounds,” Journal ofMedicinal Chemistry 1973, vol. 16, pp. 1043-1045), or a functionalequivalent of such molecules. A functional equivalent of3-amino-3-(2-nitrophenyl)propionic acid is3-amino-3(4-nitrophenyl)propionic acid. A functional equivalent ofortho-nitrophenyl-glycine is para-nitrophenyl-glycine.

In another preferred embodiment, the periodate-sensitive group comprisesa 1,2-dihydroxy moiety or a functional equivalent thereof. There arealternative systems that are equivalent to the periodate and1,2-dihydroxy system. Such systems are 1-amino-2-hydroxy systems andpolyols, carbohydrates, sugar-amino acid hybrids containing aperiodate-cleavable site. Dihydroxyethylene peptide isosters aredescribed in Suvit Thaisrivongs, Alfredo G. Tomasselli, Joseph B. Moon,John Hui, Thomas J. McQuade, Steve R. Turner, Joseph W. Strohbach, W.Jeffrey Howe, W. Gary Tarpley, and Robert L. Heinrikson, “Inhibitors ofthe protease from human immunodeficiency virus: design and modeling of acompound containing a dihydroxyethylene isostere insert with highbinding affinity and effective antiviral activity,” Journal of MedicinalChemistry 1991, vol. 34, pp. 2344-2356; Suvit Thaisrivongs, Steve R.Turner, Joseph W. Strohbach, Ruth E. TenBrink, W. Gary Tarpley, ThomasJ. McQuade, Robert L. Heinrikson, Alfredo G. Tomasselli, Joseph B. Moon,John O. Hui, W. Jeffrey Howe, “Inhibitors of the protease from humanimmunodeficiency virus: synthesis, enzyme inhibition, and antiviralactivity of a series of compounds containing the dihydroxyethylenetransition-state isostere,” Journal of Medicinal Chemistry 1993, vol.36, pp. 941-952; and Iwao Ojima, Hong Wang, Tao Wang and Edward W. Ng,“New approaches to the asymmetric synthesis of dipeptide isosteres viabeta-lactam synthon method,” Tetrahedron Letters 1998, vol. 39, pp.923-926. The synthesis of 4-amino-4-deoxy-L-threonic acid(diol-containing amino acid building block), which is anotherperiodate-sensitive compound, is described in James A. Musich and HenryRapoport, “Synthesis of Anthopleurine, the alarm pheromone fromanthopleura elegantissima,” Journal of the American Chemical Society1978, vol. 100, pp. 4865-4872.

The technology can be used for many different proteinaceous moleculesinteracting with ligands of a proteinaceous nature (multimers). In apreferred embodiment, a multimeric protein of the disclosure comprisesat least two members wherein a first member comprises a peptidecomprising the conditionally reactive group and wherein a second membercomprises a polypeptide, wherein the first and the second member bind toeach other via a region of non-covalent interaction. Preferably, themultimeric protein is a peptide-binding protein, preferably apeptide-presenting protein, and bound peptide. Preferred examples ofsuch peptide-binding proteins are proteins with SH2-SH3 domains,chaperone proteins or major histocompatibility complex molecules.Preferred chaperone proteins are heat shock proteins. Examples of theseheat shock proteins are HSP70, gp96, gp110 and calreticulin. Heat shockproteins loaded with antigens (peptides) are, for instance, used tospecifically stimulate the immune system. This specific stimulationhelps to combat diverse diseases, for instance, disorders caused by thepapillomavirus or cancer (see, for instance, Parmiani et al., 2004).

A preferred multimer is a major histocompatibility complex (MHC)molecule or a functional part, derivative and/or analogue thereof. MHCis recognized by T-cells. T-cells play a crucial role in the humanimmune system and a multitude of strategies has been developed toenhance this natural defense system and boost immunity against pathogensor malignancies. T-cells recognize MHC molecules that have bound aspecific ligand and production methods for MHC molecules that have bounda specific ligand are of substantial value. In addition, MHC moleculesare also recognized by a series of other receptors providing anadditional rationale for the production of ligand-bound MHC molecules.In addition, definition of disease-associated ligands that bind to MHCmolecules is of value for both diagnostic and therapeutic purposes.

An important use of ligand-MHC complexes is illustrated by the fact thatcomplexes of ligand-occupied MHC molecules are highly valuable tools inthe medical field to identify and quantify specific T-cell populationsand evaluate establishment of effective cellular immunity in relation todisease progression. A very large potential for MHC complexes lies inthe immune monitoring of clinical trials and approved therapeuticinterventions. In addition, MHC complexes can be applied to isolatespecific human T-cells for cellular immunotherapy against pathogens ormalignancies or to eliminate undesired T-cells from preparations forbone marrow transplantations. In addition, MHC complexes may be used toselectively eliminate undesired specific T-cell populations inT-cell-mediated diseases.

A major obstacle for the effective application of MHC molecules occupiedwith a defined ligand is the inefficiency of the current productionmethods. The stability of MHC molecules and, in particular, MHC class Imolecules, is low when antigen is not bound. Consequently, MHC moleculesare produced primarily in processes in which the ligand is bound duringthe production process. Exchange of this bound ligand for a ligand ofchoice has been used, but this process is inefficient becausedissociation of ligand is slow under mild conditions (i.e., near neutralpH and physiological salt concentration). Dissociation of bound ligandcan be promoted by exposing MHC molecules to more harsh conditions(e.g., acidic or alkaline pH), but this also leads to destabilization ofthe MHC molecule. Consequently, the technology that is now most widelyaccepted for the generation of recombinant MHC molecules is the separateproduction of a batch of ligand-occupied MHC molecules for each singleligand. This results in a very time-consuming and costly productionprocess, yielding small batches specific for only one application. (A.H. Bakker and T. N. Schumacher, “MHC multimer technology: current statusand future prospects,” Curr. Opin. Immunol. 2005, August; 17(4):428-33,Review.)

MHC molecules can be divided into two classes, MHC class I and class IImolecules. Both types of MHC molecules can bind peptides and presentthem to T-cells. Depending on the MHC molecule, the domains responsiblefor binding of the peptide have different nomenclatures. Typically, twodomains are required for specifically binding a peptide, as exemplifiedby the alpha1 and alpha2 domains of an MHC class I molecule. Thesedomains are considered a functional part of an MHC molecule. Afunctional part thus typically contains two of the domains that in anMHC molecule are involved in binding of a peptide. A natural MHCmolecule typically contains several other domains that are not directlyinvolved in binding of a peptide. Such domains typically have otherfunctions. For instance, there is a transmembrane domain or a cytosolicdomain. In addition, such domains may play a role on the formation ofthe folded conformation of the peptide-binding domains. Other domainscan, like the peptide-binding domains, be extra-cellular. All theseother domains share one characteristic. They are not directly involvedin peptide binding. Thus, for this disclosure, they may be present in apart of an MHC molecule or not, as long as the part comprises at leasttwo of the domains that are involved in peptide binding in an MHCmolecule. Preferably, the MHC molecule is a soluble MHC molecule,preferably as described in D. N. Garboczi, D. T. Hung, and D. C. Wiley,“HLA-A2-peptide complexes: refolding and crystallization of moleculesexpressed in Escherichia coli and complexed with single antigenicpeptides,” Proc. Natl. Acad. Sci. U.S.A. 1992 Apr. 15; 89(8):3429-33.

In a preferred embodiment, the MHC molecule is an MHC complex molecule.

A functional derivative of an MHC molecule is a molecule that is notderived from nature, but that shares at least a peptide-binding propertywith an MHC molecule in kind, not necessarily in amount. For instance,modified MHC molecules comprising one or more amino acid differenceswith natural MHC molecules but that retain a peptide-binding functionare functional derivatives in the context of this disclosure. Similarly,molecules comprising (part of) peptide-binding domains from two or moreMHC molecules and that are capable of binding a peptide are alsoconsidered functional derivatives. Modifications that are typicallytolerated are those that are not in the peptide-binding domains. Othermutations or modifications that are tolerated are in the variabledomains of the peptide-binding domains of MHC molecules. Suchmodifications typically alter the binding specificity of the MHCmolecule (i.e., which peptide is bound). Such modifications are,therefore, also considered functional derivatives of MHC molecules ofthe disclosure.

Several molecules share the peptide-binding properties of MHC moleculesbut have evolved to serve a different purpose in the cell. Suchmolecules are considered functional analogues of an MHC molecule of thisdisclosure. Domains that are involved in (poly)peptide binding can becombined with such domains from MHC molecules. MHC molecules orfunctional parts, derivatives and/or analogues thereof, may furthercontain other parts that are not normally associated with MHC molecules.Such other parts may, for instance, comprise labels, tags, associationand/or multimerization domains and other elements.

The technology of this disclosure can be used to specificallydestabilize ligands bound to MHC molecules, or to functional parts,derivatives and/or analogues thereof. Destabilization of the MHC-boundligands then results in the generation of ligand-free MHC moleculeswithout exposure to harsh conditions. The resulting ligand-free MHCmolecules may then be used either in the ligand-free form or may beloaded with one or multiple ligands of choice.

Thus, in a preferred aspect of this disclosure, an MHC molecule or afunctional part, derivative and/or analogue thereof, comprises a peptideantigen (also referred to as ligand) in the peptide-binding groove ofthe MHC molecule or a functional part, derivative and/or analoguethereof. The inducible attacking group or conditionally reactive groupis preferably present in the peptide antigen as this warrants release ofthe peptide antigen from the otherwise unmodified MHC molecule or afunctional part, derivative and/or analogue thereof. The resultantligand-free MHC molecules may be used directly or be loaded with one ormore other ligands.

To this end, the disclosure further provides a composition comprising amultimeric protein of the disclosure. Such a composition can be providedwith ligand to be loaded onto the MHC molecule. Thus, further providedis a major histocompatibility complex (MHC) molecule or a functionalpart, derivative and/or analogue thereof, wherein at least one of themembers is a peptide antigen in the peptide-binding groove of themolecule and wherein the peptide antigen comprises the induciblecleavable group or conditionally reactive group. The composition canalso comprise a further peptide.

In a preferred embodiment, the further peptide is an antigenic peptidecapable of binding in the peptide-binding groove of the MHC molecule,i.e., a ligand for the MHC molecule. The attacking group of the peptidemay be induced, thereby resulting in release of the peptide fragmentsfrom the multimer. If the further peptide is also present in thecomposition, this peptide can now take the place of the leavingfragments. The resultant MHC molecule or functional part, derivativeand/or analogue thereof is thereby loaded with the further peptide.Thus, the composition contains the newly loaded MHC molecule (orfunctional part, derivative and/or analogue thereof) and fragments ofthe leaving peptide.

In another aspect, the disclosure provides a method for producing an MHCmolecule or a functional part, derivative and/or analogue thereof, or anMHC molecule complex comprising a further peptide, comprising producingan MHC molecule comprising a temporary peptide having an induciblecleavable group or conditionally reactive group that, when activated,cleaves the temporary peptide into at least two smaller peptides thatexhibit reduced binding affinity for the MHC molecule. The temporarypeptide is preferably present in the peptide-binding groove of the MHCmolecule or a functional part, derivative and/or analogue of the MHCmolecule. The method preferably further comprises activating thecleavable group or conditionally reactive group.

As a result of the activation, the temporary or leaving peptide is cutinto smaller peptides (or amino acids), thereby reducing the strength ofthe noncovalent interaction in the region of noncovalent interaction.This allows the easy removal of the leaving peptide (or fragmentsthereof) from the MHC molecule or functional part, derivative and/oranalogue thereof. Removal does not require harsh conditions and, thus,only minimally interferes with the activity of the molecule, if at all.The free MHC molecule can be provided with a desired peptide. Using amethod of the disclosure, it is possible to produce large amounts of MHCmolecule having the leaving peptide. This preparation can subsequentlybe used to generate MHC molecules comprising a variety of differentligands (antigenic peptides) with a method of the disclosure. This onlyrequires activation (induction) of the cleavable group that cleaves theleaving peptide, which allows for the exchange with the desiredantigenic peptide.

Induction of the cleavable group or conditionally reactive group, thedissociation of leaving peptide fragments and association of the desiredpeptide to the MHC molecule or functional part, derivative and/oranalogue thereof, can be performed in one step. Thus, the disclosurefurther provides a method of the disclosure further comprisingincubating the MHC molecule or functional part, derivative and/oranalogue thereof, with the desired peptide under conditions thateffectively removes cleaved temporary peptide from the MHC molecule.

Further provided is a method that further comprises detecting binding ofthe desired peptide to the MHC molecule. This aspect is, for example,useful for diagnostic purposes. Binding can be detected in various ways,for instance, via T-cell receptor or antibody specific for the peptidepresented in the context of the MHC molecule. Binding is preferablydetected by detecting a label that is associated with the peptide. Thiscan be done by tagging the peptide with a specific binding molecule,such as biotin, that can subsequently be visualized via, for instance,labeled streptavidin or analogues thereof.

In a preferred embodiment, the peptide comprises the label. In this way,any peptide bound to the MHC molecule can be detected directly.Detection of binding is preferably done for screening purposes,preferably in a high throughput setting. Preferred screening purposesare screening for compounds that affect the binding of the peptide tothe MHC molecule. For instance, test peptides or small molecules cancompete with binding of the peptide to the MHC molecule. Competition canbe detected by detecting decreased binding of the peptide. A preferredmethod for detecting binding of the peptide to the MHC molecule ismeasured by means of fluorescence anisotropy. In this way, manipulationsof the sample wherein binding is performed can be reduced. Reduction ofsample manipulations is a desired property for high throughput settings.Other preferred means for detecting binding of the peptide aremonitoring radioactivity or by monitoring binding of an MHCconformation-dependent binding body, preferably an antibody or afunctional part, derivative and/or analogue thereof. Other preferredmeans include the use of a T-cell receptor specific for the combinationof the peptide, MHC molecule.

In a preferred embodiment, inhibition or enhancement of binding of thepeptide to the MHC molecule is measured. In a preferred embodiment, themethod is used for determining binding of the desired peptide in thepresence of a test or reference compound.

The disclosure further provides an MHC molecule obtainable by a methodof the disclosure. Further, the disclosure provides a compositioncomprising an MHC molecule according to the disclosure, wherein thecomposition comprises an MHC molecule complex comprising a peptidecomprising a conditionally reactive group or a derivative thereof and anMHC molecule complex comprising a further peptide.

Multimers of the disclosure may further be incorporated into even largerstructures that are further referred to as complexes of MHC multimers oras MHC tetramers, to distinguish them from the multimers of thedisclosure. Such complexes have a higher affinity for the particles andcells carrying T-cell receptors than the single MHC multimer. Suchcomplexes are, therefore, important tools in the analysis of T-cellpopulations. The disclosure thus further provides a complex comprisingat least two multimers of the disclosure or at least two MHC moleculesor functional parts, derivatives and/or analogues thereof of thedisclosure or a combination thereof. Means and methods for producingcomplexes containing two, three, four and five MHC molecules orfunctional parts, derivatives and/or analogues thereof are available inthe art. Thus, this disclosure further provides a complex comprisingtwo, three, four or five MHC molecules or functional parts, derivativesand/or analogues thereof.

In a preferred embodiment, the complexes comprise MHC molecules havingthe same T-cell receptor specificity. However, this need not always bethe case. Considering the relative ease with which MHC molecules can beprovided with different peptides using a method of the disclosure,complexes comprising two or more T-cell receptor specificities arewithin the scope of this disclosure.

Further provided is a solid surface that comprises at least twomultimers according to the disclosure, a composition according to thedisclosure or an MHC molecule or functional part, derivative and/oranalogue of the disclosure. In a preferred embodiment, the solid surfaceis provided with a complex of the disclosure, preferably a complexcomprising a single peptide, or multiple peptides associated with thesame disease or pathogen. The solid surface can be a bead. The solidsurface can be a glass or metallic surface. The surface may haveundergone pre-treatment prior to coating of the multimer, composition orcomplex of the disclosure. Such pre-treatment may include, but is notlimited to, polyacrylamide film coating as described by Soen et al.(PLoS Biology, 2003: vol. 1, pages 429-438).

Further provided is a microarray that comprises a multimer, compositionor complex of the disclosure. Means and methods for producing a(micro)array comprising an MHC molecule complex coupled to antigenicpeptide is described by Soen et al. mentioned above. The artisan isreferred to that reference for guidance as to the generation of a(micro)array of the disclosure.

The disclosure surprisingly found a novel influenza A nucleoproteinT-cell epitope in an H5N1 strain. The novel epitope interestingly has asubstantially greater immunogenicity than a classical epitope found inH5N1 strains and many other influenza A strains. Furthermore, the novelepitope is shared between H5N1 strains of the past years but is distinctfrom older influenza A strains, which makes it an epitope that is moresuitable for present and future diagnostic and therapeutic purposes thanclassic epitopes.

Provided are peptides that comprise the H5N1 epitope sequence. Thedisclosure, therefore, provides an isolated and/or synthesized peptidecomprising amino acid sequence AMDSNTLEL (SEQ ID NO:1). Further,provided is the use of an isolated and/or synthesized peptide comprisingamino acid sequence AMDSNTLEL (SEQ ID NO:1) in a vaccine. Also providedis the use of an isolated and/or synthesized peptide comprising aminoacid sequence AMDSNTLEL (SEQ ID NO:1) for the preparation of a vaccineagainst influenza. A peptide of the disclosure is, for instance,presented to the immune system of an individual in a vaccine comprisingthe peptide and an immune-enhancing agent.

In one embodiment, provided is a method for immunizing an individualagainst influenza, comprising providing the individual with an isolatedand/or synthesized peptide comprising an amino acid sequence AMDSNTLEL(SEQ ID NO:1) and optionally an immune-enhancing agent and/or a suitableadditive. “Immunizing an individual” as used herein means that theindividual develops an immune response against the peptide. Thedisclosure further provides a fusion protein comprising a peptidecomprising the amino acid sequence AMDSNTLEL (SEQ ID NO:1). Furtherprovided is a nucleic acid molecule encoding the AMDSNTLEL (SEQ ID NO:1)peptide or the fusion protein.

In one embodiment, provided is an MHC molecule hereof, comprising anisolated and/or synthesized peptide comprising amino acid sequenceAMDSNTLEL (SEQ ID NO:1). In one embodiment of the disclosure, the MHCmolecule is for the detection of epitope-specific T-cells. The influenzais preferably H5N1. An influenza virus to diagnose is preferably acurrent variant of an influenza virus.

In one embodiment, provided is the use of an MHC molecule according tothe disclosure for the preparation of a composition for diagnosinginfluenza. In a further embodiment, provided is a method for diagnosinginfluenza in an individual, comprising providing a blood sample of theindividual with an MHC molecule according to the disclosure andanalyzing binding of the MHC molecule to a cell in the blood sample. Thecell in the blood sample is preferably a T-cell. In a preferredembodiment, a method for diagnosing influenza in an individual furthercomprises detecting a T-cell response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Photocleavage strategy: Panel A, structure of I, the unmodifiedinfluenza A 58-66 epitope ((“Flu peptide 1”) sequence GILGFVFTL (SEQ IDNO:2); Panel B, photocleavage reaction; and Panel C, the structure ofIII is the T8*-modified photocleavable influenza A 58-66 epitope((“photocleavable Flu analog 6”) sequence GILGFVF*L (SEQ ID NO:4)where * is 3-amino-3-(2-nitrophenyl) propionic acid).

FIG. 1B. Oxidative periodate cleavage strategy: Panel A, cleavagereaction; Panel B, building blocks; Panel C, structure of periodatecleavable flu peptide (sequence GI*GFVFTL (SEQ ID NO:6) where * is adipeptide isostere).

FIGS. 2A and 2B. Biochemical analysis of UV-induced peptide exchange:FIG. 2A, MHC multimers containing a UV-sensitive ligand are sensitive toUV exposure; and FIG. 2B, UV-sensitive MHC multimers that are exposed toUV can be stabilized by addition of MHC-binding peptides.

FIG. 3. MHC tetramers generated from UV-sensitive MHC multimers stain anantigen-specific CTL clone. UV-sensitive MHC multimers were exposed toUV in the presence of the indicated peptides and were converted totetrameric complexes of MHC multimers. Thus generated tetramerscontaining either the HY (SMCY) 311-319 peptide (lower left panel) orMART I 26-35 (A2L mutant) peptide (lower right panel) were used to staina MART I 26-35-specific CTL clone. As a control, the same clone wasstained using classical MHC class I tetramers containing the sameepitopes (top panels).

FIG. 4. MHC tetramers generated from UV-sensitive MHC multimers stainantigen-specific peripheral blood T-cells. UV-sensitive MHC multimerswere exposed to UV in the presence of the indicated peptides and wereconverted to MHC tetramers. Thus generated tetramers containing eitherthe CMV pp65 495-503 peptide (lower left panel) or influenza A matrix58-66 peptide (lower right panel) were used to stain peripheral bloodmononuclear cells from a donor with both CMV pp65 495-503 and influenzaA matrix 58-66-specific CD8+ T-cells. As a control, the same PBMCs werestained using classical MHC class I tetramers containing the sameepitopes (top panels).

FIG. 5. MHC tetramers generated from UV-sensitive MHC tetramers stainantigen-specific peripheral blood T-cells. UV-sensitive MHC tetramerswere exposed to UV in the presence of the indicated peptides and wereused to stain antigen-specific T-cells without further purification. MHCtetramers containing either the CMV pp65 495-503 peptide (second leftpanel), or MART I 26-35 (A2L mutant) or HY (SMCY) 311-319 peptide (tworight-most panels) were used to stain peripheral blood mononuclear cellsfrom a donor with CMV pp65 495-503 CD8+ T-cells. As a control, the samePBMCs were stained using classical MHC class I tetramers containing theCMV pp65 495-503 peptide (left panel).

FIG. 6. Gel filtration profile of photolabile peptide/HLA-A2.1 complexbefore irradiation (1), after irradiation without rescue (2), afterirradiation in the presence of peptide FLPSDC*FPSV (SEQ ID NO:5) whereC* is labeled with a tetramethylrhodamine dye (3). Left panel: UVdetection at 230 nm; Right panel: Fluorescence detection (excitation 530nm, emission 550 nm).

FIG. 7. Fluorescence anisotropy screen. Peptide-free MHC molecules aregenerated by cleavage of the conditional ligand. The binding of afluorescent epitope is monitored by measurement of fluorescenceanisotropy or fluorescence detection. By this method, peptidic ornon-peptidic ligands that interfere with or facilitate such binding canbe identified.

FIG. 8A. Diol-based chemocleavable conditional peptide (sequenceGILGFVF*L (SEQ ID NO:7) where * is 4-amino-2,3-dihydroxybutanoic acid)and the corresponding cleavage products. The diol-containing unit isboxed.

FIG. 8B. Oxidative periodate-mediated peptide cleavage. Panel A,cleavage reaction; Panel B, building blocks compatible with solid phasepeptide synthesis (protected dihydroxy amino acid 10 and protecteddipeptide isostere 11); and Panel C, deprotected periodate-cleavable flupeptide 12 containing diol dipeptide isostere 11.

FIG. 9. Cleavage of MHC-binding peptides by NaIO4. Peptide variants ofthe HLA-A2.1-binding M1 peptide with a diol-containing building block atposition 8 (FIG. 8A) or position 4 (FIG. 8B) were produced by chemicalsynthesis. Peptides were then analyzed by LC-MS, prior to (bottom,sequence GILGFVF*L (SEQ ID NO:7) where * is4-amino-2.3-dihydroxybutanoic acid) or following (top) a one-hourexposure to 1 mM NaIO4.

DETAILED DESCRIPTION Examples

Methods:

Production of MHC Multimers and Complexes of MHC Multimers (MHCTetramers):

MHC class I complexes (MHC multimers) were prepared as previouslydescribed with minor modifications.⁽¹⁾ HLA-A2.1-peptide multimers weregenerated with the following three peptides: Influenza-A matrix 58-66(sequence GILGFVFTL (SEQ ID NO:2)) and the two influenza A matrix 58-66variants GIL*FVFTL (SEQ ID NO:3 and GILGFVF*L (SEQ ID NO:4) where * is3-amino-3-(2-nitrophenyl)propionic acid. MHC class I-peptide multimerswere subsequently purified, biotinylated by BirA, purified and stored at−20° C. in 16% glycerol.

UV-Induced Peptide Liberation and Peptide Exchange:

MHC multimers or, where indicated, tetrameric complexes of MHC multimerscontaining the wild-type influenza A matrix 58-66 epitope, or the G4* orT8* variants of this epitope, were exposed for one huor to UV (CAMAG,366 nm) in 20 mM Tris-HCl, pH 7.0/150 mM NaCl/0.5 mM dithiothreitol(DTT) in the presence or absence of MHC class I binding peptides.Subsequently, the complex was exposed to 37° C. for 15-45 minutes toinduce dissociation of peptide-free MHC class I complexes.⁽²⁾ Sampleswere then analyzed by gel filtration chromatography to determine MHCdissociation, or were incubated with phycoerythrin-labeled streptavidinto generate tetrameric complexes of MHC multimers (MHC tetramers). MHCtetramers were purified by gel filtration chromatography and stored at−20° C. in 16% glycerol until further use.

MHC Tetramer Staining:

Thawed peripheral blood mononuclear cells (PBMC) samples and CTL cloneswere incubated for five minutes with PE-labeled MHC tetramers at 37° C.,FITC-labeled anti-CD8 antibody was added and incubation was continuedfor 15 minutes at room temperature. Prior to FACS analysis, cells werestained with propidium iodide to be able to gate out dead cells. Sampleswere analyzed by flow cytometry using a FACScalibur and CellQuestsoftware (Becton Dickinson). Forward and side scatter parameters wereused to define lymphocyte populations.

Results:

Formation of Peptide-MHC Multimers that Dissociate at Will.

To test the feasibility of generating MHC multimers of which the boundpeptide could be liberated at will, we generated HLA-A2.1 multimers witheither the wild-type influenza A matrix 58-66 epitope, or two variantsof this epitope in which either amino acid 4 or amino acid 8 wasreplaced by the UV-sensitive beta-amino acid 3 (FIG. 1). MHC class Imultimer formation was efficient for all three peptides and themultimers formed were purified by gel filtration chromatography. Toassess whether the resulting peptide-MHC multimers could be induced todissociate, the three types of multimers were exposed to UV, incubatedat 37° C. to induce dissociation of remaining peptide-free MHC class Imolecules and then analyzed by gel filtration chromatography.

Whereas, the peptide-MHC multimer containing the parental influenza Aepitope is fully insensitive to UV exposure (FIG. 2A top panel),exposure of MHC class I multimers containing either the G4* (data notshown) or the T8* epitope (FIG. 2A bottom panel) leads to a substantialreduction in the amount of MHC complex recovered. Furthermore, theremaining material most likely consists, at least in part, of free MHCclass I heavy chains rather than peptide-MHC class I multimers, assuggested by the fact that the elution time of remaining material isslightly greater than that of the starting material.

Protection of MHC Class I Dissociation by Addition HLA-A2.1-BindingPeptides.

To assess whether the addition of MHC class I binding ligands couldprotect the dissociation of UV-sensitive MHC complexes upon UV exposure,the same reactions were performed either in the presence of one of twodifferent HLA-A2.1-binding peptides (HY 311-319; MART I 26-35 (A2Lmutant), or the HLA-A3-binding peptide GP100 (614-622). Addition ofeither of the three peptides to MHC class I molecules containing theparental influenza A epitope does not affect the recovery of the MHCclass I multimer, regardless of whether the multimer is exposed to UV,consistent with the notion that this parental MHC multimer is stableunder both conditions (data not shown). Importantly, while addition ofthe HLA-A3-binding peptide that is not expected to interact withHLA-A2.1 does not lead to a substantial increase in the recovery of theUV-sensitive G4* (not shown) or T8* peptide-containing (FIG. 2B) MHCmultimer, the addition of either HLA-A2.1-binding peptide leads to ahighly increased recovery (FIG. 2B). These data are consistent with thenotion that the peptide-free MHC molecules that are generated uponexposure of the G4*- or T8*-containing MHC multimer to UV canefficiently bind known HLA-A2.1 ligands but not a control peptide.

Functional Tetrameric Complexes of MHC Multimers Generated fromUV-Sensitive MHC Multimers.

To directly establish whether the UV-sensitive MHC class I multimersthat had been protected by addition of HLA-A2.1 ligands had bound theseligands, MHC multimers generated in the presence of either the MART I26-35 (A2L mutant), influenza A matrix 58-66, HY (SMCY) 311-319, or CMVpp65 495-503 peptide were purified and converted to tetrameric complexesof MHC multimers. The resulting MHC tetramers were subsequently used tostain either a MART I-specific T-cell clone (FIG. 3) or peripheral bloodmononuclear cells from a donor with both CMV pp65 495-503 and influenzaA matrix 58-66-specific CD8+ T-cells (FIG. 4). In all cases tested, MHCtetramers generated following peptide exchange bind antigen-specificT-cells with equal sensitivity and specificity as compared toconventional MHC tetramers. These experiments provide formal proof thatMHC multimers generated by peptide exchange are structurallyindistinguishable from conventional peptide-MHC multimers and can beused to probe pMHC-TCR interactions, in this case followingoligomerization.

Functional MHC Tetramers Generated from UV-Sensitive MHC Tetramers.

To establish whether peptide exchange could also be performed onUV-sensitive tetrameric complexes of MHC class I multimers,T8*-containing MHC multimers were converted to tetrameric complexes andsubsequently exposed to UV in the presence of either the CMV pp65495-503, MART I 26-35 (A2L mutant), or HY (SMCY) 311-319 peptide. Theresulting tetrameric MHC complexes were subsequently used withoutfurther purification to stain peripheral blood mononuclear cells of aCMV-positive donor. Remarkably, MHC tetramers generated by UV exposureof T8*-containing MHC tetramers in the presence of the CMV pp65 495-503epitope detect CMV-specific T-cells at an equal frequency and withsimilar intensity as conventional CMV pp65 495-503-specific MHCtetramers (FIG. 5). The specificity of this binding is underscored bythe fact that MHC tetramers prepared in parallel reactions with eitherthe MART I 26-35 (A2L mutant), or HY (SMCY) 311-319 peptide do not showmeasurable binding to CD8+ lymphocytes of this donor.

Other Aspects of MHC Exchange Technology.

UV exchange technology was used to generate MHC molecules that arereceptive to binding of ligands that carry a label. MHC exchangereactions were carried out in the presence of a peptide ligand that hadbeen labeled with a tetramethylrhodamine dye. As shown in FIG. 6,subsequent analysis of these reactions reveals that this technology canbe used to allow binding of a labeled ligand, in this case a fluorescentpeptide. Consequently, MHC exchange technology can also be used toscreen for compounds (e.g., peptides and other small molecules) that canenhance or interfere with such binding. The principle of such a screen,here exemplified using fluorescence anisotropy, is outlined in FIG. 7.

Modification of known MHC-binding peptides allows the generation ofligands that are sensitive to chemical cleavage, variants of theinfluenza A matrix 58-66 peptide were produced in which adiol-containing building block is incorporated. An example of such amodified ligand is given in FIG. 8A. Exposure of such modified ligandsto periodate leads to cleavage of these ligands, as exemplified in FIG.9.

Discussion:

The current data describe a novel approach for the generation of MHCcomplexes that are occupied with a peptide of choice. The mainlimitation in the production of such complexes has been the instabilityof peptide-free MHC molecules. Consequently, the technology that is nowwidely accepted for the generation of recombinant MHC molecules is theseparate production of a batch of ligand-occupied MHC molecules for eachsingle ligand. This results in a very time-consuming and costlyproduction process, yielding small batches specific for only oneapplication. Here, we demonstrate that MHC molecules occupied by aligand of choice can be generated by the selective release of apreviously bound ligand, by exposure to conditions that do not directlyaffect the stability of the MHC complex itself.

In the current set of experiments, dissociation of MHC-bound ligand wasachieved through the use of a light-sensitive peptide variant. However,it is apparent that such dissociation may equally well be achievedthrough the use of peptide variants that are sensitive to other definedconditions. In particular, the development of peptide-MHC complexesusing peptide variants that are sensitive to chemical cleavage appearuseful in this respect. In addition, dissociation may also be achievedwithout peptide cleavage, by inducing a reduced affinity of the boundligand for MHC through chemical- or light-induced modification.Furthermore, while the approach for ligand exchange has here beendeveloped for MHC class I molecules, this approach should be equallyuseful to prepare ligand-occupied MHC class II molecules ornon-classical MHC molecules (exemplified by CD1 and Qa1 molecules).

Recombinant MHC molecules generated through chemical- or light-inducedpeptide release will be of substantial use to generate the vastcollections of MHC complexes that are currently used in clinical andpreclinical research. In addition, the ability to generate MHC ligandsoccupied by a desired ligand through simple exchange should greatlyfacilitate efforts to produce GMP grade MHC molecules that can be usedfor selective antigen-specific T-cell depletion or enrichment. Finally,the ability to perform peptide exchange on preformed MHC complexes formsa viable strategy to generate microarrays of MHC complexes occupied withlarge collections of peptide antigens. Such MHC microarrays may formuseful tools for the high throughput analysis of the antigen-specificT-cell repertoire.⁽³⁾

REFERENCES

-   1. Altman J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. G.    McHeyzer-Williams, J. I. Bell, A. J. McMichael and M. M. Davis.    Phenotypic analysis of antigen-specific T-lymphocytes. Science    274:94-96 (1996).-   2. Schumacher T. N. M., M.-T. Heemels, J. J. Neefjes, W. M.    Kast, C. J. M. Melief, and H. L. Ploegh. Direct binding of peptide    to empty MHC class I molecules on intact cells and in vitro. Cell    62:563-567 (1990).-   3. Soen Y., D. S. Chen, D. L. Kraft, M. M. Davis M M, and P. O.    Brown. Detection and Characterization of Cellular Immune Responses    Using Peptide-MHC Microarrays. PLoS Biol. 1:E65: pp. 429-438 (2003).-   4. Parmiani G., A. Testori, M. Maio, C. Castelli, L. Rivoltini, L.    Pilla, F. Belli, V. Mazzaferro, J. Coppa, R. Patuzzo, M. R.    Sertoli, A. Hoos, P. K. Srivastava and M. Santinami. Heat shock    proteins and their use as anticancer vaccines. Clinical Cancer    Research 10:8142-8146 (2004).

What is claimed is:
 1. A major histocompatibility complex (MHC) moleculecomprising a temporary peptide in the peptide binding groove, whereinthe temporary peptide comprises at least one reactive group that, whenactivated by a chemical or physical signal, cleaves the temporarypeptide into at least two peptides, which each exhibit reduced bindingaffinity for the MHC molecule with respect to the uncleaved peptide,wherein the reactive group is a light-sensitive or a periodate-sensitivegroup.
 2. The MHC molecule of claim 1, wherein the light-sensitive groupcomprises 3-amino-3-(2-nitrophenyl)propionic acid.
 3. The MHC moleculeof claim 1, wherein the periodate-sensitive group comprises a1,2-dihydroxy moiety.
 4. A composition comprising: the MHC molecule ofclaim
 1. 5. A major histocompatibility complex (MHC) molecule producedby a method comprising: producing an MHC molecule comprising a temporarypeptide having at least one reactive group that, when activated, cleavesthe temporary peptide into at least two smaller peptides that exhibitreduced binding affinity for the MHC molecule, wherein the reactivegroup(s) is/are a light-sensitive group or a periodate-sensitive group.6. A composition comprising: the MHC molecule of claim 5, wherein thecomposition comprises a major histocompatibility complex (MHC)-moleculecomplex comprising a peptide comprising a reactive group, wherein thereactive group is a light-sensitive group or a periodate-sensitivegroup, and an MHC molecule complex comprising a further peptide.
 7. Acomplex comprising: at least two MHC molecules of claim
 1. 8. A solidsurface comprising: the MHC molecule of claim 5.